The present invention, in some embodiments thereof, relates to sensing and, more particularly, but not exclusively, to systems and methods which can be utilized, for example, for real-time simultaneous detection of a variety of samples and/or for real-time detection of redox-reactive moieties such as, for example, oxidizing moieties produced by metabolites. The systems and methods described herein can be utilized, for example, for monitoring and/or analyzing metabolic activity of cells, and hence in various diagnostic and/or therapeutic applications.
Metabolism is defined as the totality of biochemical processes in living organisms that either produce or consume energy. Metabolic processes regulate cells to grow or die, reform their structures, and respond to their environments. Abnormal metabolic reactions disturb normal physiology and lead to severe tissue dysfunction, and are linked to many diseases.
Cancer is an example of a common human disease with metabolic perturbations. Altered cellular metabolism is a hallmark of cancer, contributing to malignant transformation and to the initiation, growth, and maintenance of tumors. Thus, for example, studies have shown that altered glucose metabolism promotes cancer development, and that cancer cells consume much more glucose and secrete much more lactate than normal tissue.
Understanding the complex networks associated with cancer metabolism for monitoring thereof have therefore been recognized as desirable for distinguishing metabolic significances of cancers, estimating the effectiveness of therapies, and facilitating personalized treatments. See, for example, Munoz-Pinedo et al. Cell Death Dis 2012, 3: e248; and Griffin and Shockcor, Nature reviews Cancer 2004, 4(7): 551-561.
Several methodologies have been used heretofore for monitoring metabolic activities of cells. The most prevalent are mass spectrometry (MS) techniques linked with a separation method such as gas (GC) or liquid (LC) chromatography. In MS, species are ionized and separated based on their mass-to-charge ratio. MS is sensitive within physiological concentration ranges of metabolites, but results are obtained in endpoint fashion, ceasing metabolic activity of samples to collect data, rather than in real-time. In addition, this methodology requires sample preprocessing, rendering it incompatible with direct testing of biosamples such as blood or serum. Alternative separation methods for MS include electrospray ionization (ESI), which improves preprocessing, and nanostructure-initiator MS (NI-MS), which allows direct detection of physiological solutions. See, for example, Shulaev V. Metabolomics technology and bioinformatics. Brief Bioinform 2006, 7(2):128-139; and Northen et al. Nature 2007, 449(7165): 1033-U1033.
For real-time sensing with multiplex profiling in physiological samples, methodologies combining electrochemical and fluorescent sensing techniques have been sought for. Enzyme-reactive electrochemical sensors combining H2O2-detecting electrodes with enzyme-modified membranes to convert metabolites to H2O2 for real-time sensing have been developed [Pörtner R. Animal cell biotechnology: methods and protocols, 2nd edn. Humana Press: Totowa, N.J., 2007]. A fluorescent sensor with embedded fluorophores for detecting O2 consumption and pH change of biosamples in real time has also been developed [Marx V. Nature 2013, 494(7435), p. 131].
WO 2012/137207 describes a method of measuring a metabolic activity of a cell, effected by independently measuring in an extracellular environment of the cell, time-dependent acidification profiles due to secretion of non-volatile soluble metabolic products; non-volatile soluble metabolic products and volatile soluble metabolic products; and volatile soluble metabolic products, and uses of such a method for diagnosing and monitoring disease treatment.
Recent developments in microfluidic technology and nanotechnology have also been exploited for supersensitive real-time detection of micro-volume metabolites. Microfluidic devices which separate microlevels of metabolites in solution using electrophoresis [Garcia-Perez et al., Journal of Chromatography A 2008, 1204(2): 130-139; Garcia and Henry Anal Chim Acta 2004, 508(1): Wang et al. Anal Chim Acta 2007, 585(1): 11-16; et al. Analyst 2009, 134(3): 486-492; and Vlckova and Schwarz J Chromatogr A 2007, 1142(2): 214-221] or liquid chromatography [Wang L et al. J Microelectromech S 2008, 17(2): 318-327; Lin et al. Anal Chem 2008, 80(21): 8045-8054], have been described. Currently used microfluidic chips, however, require coupling to other detection techniques and thus require preprocessing [Kraly et al. Anal Chim Acta 2009, 653(1): 23-35].
Electrochemical, photochemical, and antibody/enzyme-functionalized nanowire sensors have also been described for detecting target metabolites. See, for example, Ramgir et al. Small 2010, 6(16): 1705-1722; and Peretz-Soroka et al. Nano Lett 2013, 13(7): 3157-3168.
Antibody/enzyme nanowire FET devices which target metabolites via binding affinity have been disclosed in, for example, Lu et al. Bioelectrochemistry 2007, 71(2): 211-216; Patolsky et al. Nanowire-based biosensors. Anal Chem 2006, 78(13): 4260-4269; and Yang et al. Nanotechnology 2006, 17(11): S276-S279.
Electrochemically-sensitive nanowire sensors for detecting metabolites by oxidative reactions have been disclosed in, for example, Lu et al. Biosens Bioelectron 2009, 25(1): 218-223; Krivitsky et al. Nano letters 2012, 12(9): 4748-4756; Shao et al. Adv Funct Mater 2005, 15(9): 1478-1482; Su et al. Part Part Syst Char 2013, 30(4): 326-331; and Tyagi et al. Anal Chem 2009, 81(24): 9979-9984.
Semiconducting nanowires are known to be extremely sensitive to chemical species adsorbed on their surfaces. For a nanowire device, the binding of a charged analyte to the surface of the nanowire leads to a conductance change, or a change in current flowing through the wires. The 1D (one dimensional) nanoscale morphology and the extremely high surface-to-volume ratio make this conductance change to be much greater for nanowire-based sensors versus planar FETs (field-effect transistors), increasing the sensitivity to a point that single molecule detection is possible.
Nanowire-based field-effect transistors (NW-FETs) have therefore been recognized in the past decade as powerful potential new sensors for the detection of chemical and biological species. See, for example, Patolsky et al., Analytical Chemistry 78, 4260-4269 (2006); Stern et al., IEEE Transactions on Electron Devices 55, 3119-3130 (2008); Cui et al., Science 293, 1289-1292 (2001); Patolsky et al. Proceedings of the National Academy of Sciences of the United States of America 101, 14017-14022 (2004), all being incorporated by reference as if fully set forth herein.
Studies have also been conducted with nanowire electrical devices for the simultaneous multiplexed detection of multiple biomolecular species of medical diagnostic relevance, such as DNA and proteins [Zheng et al., Nature Biotechnology 23, 1294-1301 (2005); Timko et al., Nano Lett. 9, 914-918 (2009); Li et al., Nano Lett. 4, 245-247 (2004)].
Generally, in a NW-FET configuration, the gate potential controls the channel conductance for a given source drain voltage (VSD), and modulation of the gate voltage (VGD) changes the measured source-drain current (ISD). For NW sensors operated as FETs, the sensing mechanism is the field-gating effect of charged molecules on the carrier conduction inside the NW. Compared to devices made of micro-sized materials or bulk materials, the enhanced sensitivity of nanodevices is closely related to the reduced dimensions and larger surface/volume ratio. Since most of the biological analyte molecules have intrinsic charges, binding on the nanowire surface can serve as a molecular gate on the semiconducting SiNW [Cui et al., 2001, supra].
U.S. Pat. No. 7,619,290, U.S. Patent Application having publication No. 2010/0022012, and corresponding applications, teach nanoscale devices composed of, inter alia, functionalized nanowires, which can be used as sensors.
Clavaguera et al. disclosed a method for sub-ppm detection of nerve agents using chemically functionalized silicon nanoribbon field-effect transistors [Clavaguera et al., Angew. Chem. Int. Ed. 2010, 49, 1-5].
SiO2 surface chemistries were used to construct a ‘nano-electronic nose’ library, which can distinguish acetone and hexane vapors via distributed responses [Nature Materials Vol. 6, 2007, pp. 379-384].
U.S. Patent Application having Publication No. 2010/0325073 discloses nanodevices designed for absorbing gaseous NO. WO 2011/000443 describes nanodevices which utilize functionalized nanowires for detecting nitro-containing compounds.